The potency of luliconazole against clinical and environmental Aspergillus nigri complex.

Background and Objectives
Black Aspergillus strains including, Aspergillus niger and A. tubingensis, are the most cause of otomycosis with worldwide distribution. Although, amphotericin B was a Gold standard for the treatment of invasive fungal infection for several decades, it gradually replaced by fluconazole and /or voriconazole. Moreover, luliconazole, appears to offer the best potential for in vitro activity against black Aspergillus strains. The aim of the present study was to compare the in vitro activity luliconazole, with commonly used antifungals against clinical and environmental strains of black Aspergillus.


Materials and Methods
Sixty seven (37 clinical and 30 environmental) strains of black Aspergillus were identified using morphological and molecular technique (β-Tubulin gene). In addition, antifungal susceptibility test was applied according to CLSI M38 A2. The results were reported as minimum inhibitory concentration (MIC) or minimum effective concentration (MEC) range, MIC50 or MEC50, MIC90 or MEC90 and MIC geometric (GM) or MECGM.


Results
Aspergillus niger was the common isolate followed by, A. tubingensis in both clinical and environmental strains. The lowest MIC range, MIC50, MIC90, and MICGM was attributed to luliconazole in clinical strains. The highest resistant rate was found in amphotericin B for both clinical (86.5%) and environmental (96.7%) strains whereas 54.1% of clinical and 30% of environmental isolates were resistant to caspofungin. Clinical strains of Aspergillus were more sensitive to voriconazole (86.7%) than environmental strains (70.3%). On the other hand, 83.8% of clinical and 70% of environmental isolates were resistant to posaconazole.


Conclusion
Luliconazole versus amphotericin B, voriconazole, posaconazole and caspofungin is a potent antifungal for Aspergillus Nigri complex. The in vitro extremely antifungal efficacy against black Aspergillus strains of luliconazole, is different from those of other used antifungals.

While, amphotericin B was a Gold standard in the first-line treatment of invasive fungal infections for several decades (9), it has been replaced by several new antifungals including, voriconazole, posaconazole and caspofungin (10,11). Voriconazole was presented as the primary therapy for invasive pulmonary aspergillosis in a clinical trials (12). Further studies have shown that posaconazole is a useful antifungal for invasive fungal infection including aspergillosis (13). On the other hand, during 2-3 last decades, caspofungin was developed to improve the prognosis of invasive aspergillosis (14).
Invasive Aspergillus infections are one of the life threatening human disease. On the other hand, some species of Aspergillus have inherent resistance to some antifungal agents (29). Moreover, some species have raised minimum inhibitory concentration (MIC) against specific antifungals. As a results, infection prevention consultant and the best choice antifungal are common clinical challenges.
The aim of the present study was to compare the in vitro activity of a novel antifungal agent, luliconazole, with amphotericin B, voriconazole, posaconazole and caspofungin against clinical and environmental strains of black Aspergillus. Furthermore, the potency of each antifungal against clinical and environmental isolates was compared.

MATERIALS AND METHODS
Fungal isolates. Thirty seven clinical isolates of black Aspergillus strains were previously isolated from otomycosis samples, identified based on morphology characteristics and preserved at Medical Mycology laboratory affiliated to Ahvaz Jundishapur University of Medical Sciences. This project was approved by the ethical committee of Ahvaz Jundishapur University of Medical Sciences (IR.AJUMS. REC.1396.1066).
Environmental strains of black Aspergillus (30 strains) were trapped from airborne spores using Sabouraud dextrose agar (SDA) (BioLife, Italia) plates. Primary screening of black Aspergillus strains was applied based on macroscopic (Black colony) and microscopic morphology. All strains (clinical and environmental) were subcultured on SDA and re-identified using molecular tests. DNA extraction. All strains (clinical and environment isolates) were subcultured on SDA plates and incubated at 29ºC for 24-48 hours. Mycelia were collected in cryo-tubes containing 300 µL lysis buffer and 0.46 g glass beads and kept at 4ºC for 72 hours. The tube contents were homogenized using a Speed-Mill PLUS Homogenizer (Analytikjena, Germany) for 6 minutes (3 cycles) and boiled at 100ºC for 20 minutes. 300 µL of sodium acetate (3M) was added to each tube and stored at -20ºC for 10 minutes. Supernatants were removed after a centrifugation at 12000 rpm for 10 minutes. DNA was purified using phenol-chloroform-isoamyl alcohol (Merck, Germany) according to a protocol devised by Makimura et al. (30). Finally purified DNA was preserved at -20ºC for further tests.

Molecular identification. β-Tubulin gene was
used for the molecular detection of strains using primers pair, βt2a (forward), 5' GGTAACCAAATC-GGTGCTGCTTTC 3' and βt2b (reverse) 5' ACCCT-CAGTGTAGTGACCCTTGGC 3' (31). PCR products subjected for sequence analysis and then sequences were manually verified by MEGA6 software package (https://www.megasoftware.net/) and aligned using the CLUSTALW algorithm. All sequences were compared to reference sequences in the Gen-Bank (NCBI) and CBS database via the nucleotide BLAST™ algorithm to obtain a definitive identification (similarity values ≥ 99%). Finally, all nucleotide sequences representative were deposited in the Gen-Bank database.
Statistical analysis. The Chi-squared test using the Social Science Statistics software (Online) was applied to determine the significant between variables and P value < 0.05 is considered as significance level.

Morphological identification
Caspofungin was significantly more effective against environmental than clinical strains (P = 0.048) of black Aspergillus strains. However, the inhibitory effect of amphotericin B, posaconazole and voriconazole was similar against both tested strains (clinical and environmental) (amphotericin http://ijm.tums.ac.ir B, P=0.147; voriconazole, P=0.109; posaconazole, P=0.178). When we compared the effect antifungals against A. niger and A. tubingensis strains, it found that caspofungin was more effective on A. niger with environmental sources than clinical strains (P=0.0482). Whereas, the effect of other antifungals against both species was not significant.
Our results showed that 32 (86.5%) of clinical strains were resistant to 2, 3 or 4 antifungals, 2 (5.4%) isolates were resistant to one antifungal and 3 (8.1%) isolates were fully susceptible to all antifungals (Table 4). Two strains of A. tubingensis, one A. niger and one black Aspergillus strains were resistant to all antifungals (except luliconazole). On the other hand, 21 (70%) of environmental strains were resistance to 2 -4 antifungals and only 30% of strains were resistance to one antifungals (Table 5). Two strains of A. niger and one A. tubingensis were resistant to all antifungals (except luliconazole).

DISCUSSION
Aspergillus strains isolated from clinical and air borne samples were identified using classical morphological features and molecular methods. In the present study, A. tubingensis, A. luchuensis and A. piperis were identified as the cryptic species of A. niger sensu lato by the sequence analysis of β-tubulin gene. Several reports have shown that A. niger is generally as common causative agent of otomycosis and one of the most important agent for invasive aspergillosis (20,22,26,(39)(40)(41). However, this species cannot be reliably detected from other cryptic members of Aspergillus section Nigri using conventional morphological methods. Molecular tools with sequence-based techniques such as partial sequence of the β-tubulin gene are presented as the most valuable method for A. niger Nigri species assignment (4,21). These molecular techniques are indicating that this species comprises 19 cryptic species (4,16,21) with more prevalence of A. niger sensu stricto and A. tubingensis (16,42).
Our results showed that, although the luliconazole MIC ranges for strains were extremely low, this range for environmental strains (0.00781-0.00049 μg/ml) was lower than clinical strains (0.125 -0.00024 μg/ ml). As shown in Table 5, only five clinical strains (A. niger sensu stricto, 4 isolates and A. tubingensis, 1 isolate) have a MIC = 0.125 μg/ml. 30/30 (100%) of    and clinical strains were 0.00195 and 0.00295 μg/ml, respectively. Some studies have shown a high efficacy of luliconazole against dermatophytes and onychomycosis agents both in vivo and in vitro (1,2,7,8,43). Furthermore, recently a few studies examined the potency of luliconazole against different species of Candida, A. fumigatus, A. terreus and Fusarium species (5,6,44,45). However, the potency profile of luliconazole against A. niger complex is unknown. There are the limited data in in vitro efficacy of caspofungin against black Aspergillus strains from clinical and environmental sources. While, the clinical and environmental strains had the same MIC ranges for caspofungin, the resistant to antifungal showed the clear differences between clinical and environmental strains (P = 0.048), where the clinical isolates showed higher resistant rate than the environmental strains. In a report by Badali et al. only 6.1% of environmental strains of A. niger were resistant to caspofungin and all clinical isolates ranged at 0.008 -0.063 μg/ml (21). In agree with our study Araujoa et al., revealed significantly higher MIC values to caspofungin in the case of non-fumigatus clinical than environmental strains (46).
The in vitro activities of posaconazole, voriconazole, and amphotericin B against clinical Aspergillus strains have been reported by Arikan et al. (10). They reported that voriconazole was the most active antifungal against A. niger. Comparable to our results, voriconazole was more potent than the other tested antifungals (with exception luliconazole) against both clinical and environmental strains. Similar to our study, Hashimoto et al., showed no remarkable differences between the MIC distribution rate of voriconazole against clinical and environmental isolates (15). Furthermore, all tested A. niger (environment and clinical isolates) were susceptible to both amphotericin B and voriconazole in Misra et al., research (47). Aspergillus tubingensis resistant strains to amphotericin B was very common both in environment and clinical settings, followed by posaconazole, caspofungin, and voriconazole. However, the resistant rate to amphotericin B was lower among environmental than clinical strains. Hashimoto et al. finding suggests that A. tubingensis is intrinsically resistant to azole antifungals (15). Antifungal susceptibility testing of our A. tubingensis strains revealed 90.9% and 53.8% of clinical and environmental isolates were resistant to posaconazole.

CONCLUSION
In conclusion, luliconazole versus amphotericin http://ijm.tums.ac.ir B, voriconazole, posaconazole and caspofungin is a potent antifungal for Aspergillus Nigri complex. The in vitro extremely antifungal efficacy against black Aspergillus strains of luliconazole, is different from those of other used antifungals. The MIC range, MIC 50 , MIC 90 and MICGM of luliconazole against black Aspergillus strains were the lowest among the representative tested antifungals. These results suggest luliconazole can be a viable option for the treatment of infections due to black Aspergillus strains and should be further investigated in vivo.